Autocrine/Paracrine Determinants of Strain-activated Brain Natriuretic Peptide Gene Expression in Cultured Cardiac Myocytes

The application of mechanical strain leads to activation of human brain natriuretic peptide gene promoter activity, a marker of hypertrophy, in cultured neonatal rat ventricular myocytes. We have used a combination of transient transfection analysis and reverse transcriptase-polymerase chain reaction to examine the role of locally produced factors in contributing to this activation. Conditioned media from strained, but not static, cultures led to a dose-dependent increase in human brain natriuretic peptide gene promoter activity. This increase was completely blocked by losartan or BQ-123, implying a role for angiotensin and endothelin as autocrine/paracrine mediators of the response to strain. Inclusion of the same antagonists in the cultures themselves led to only partial inhibition (∼60%), whereas inclusion of exogenous endothelin or angiotensin II resulted in amplification of the strain response. Angiotensin II and endothelin appear to be arrayed in series in the regulatory circuitry; the angiotensin response was blocked by BQ-123, whereas the endothelin response was unaffected by losartan. Mechanical strain was also shown to stimulate expression of the endogenous angiotensinogen, angiotensin-converting enzyme, and endothelin genes in this system. Collectively, these data indicate that locally generated angiotensin II and endothelin, acting in series, play an important autocrine/paracrine role in mediating strain-dependent activation of cardiac-specific gene expression. The application of mechanical strain leads to activation of human brain natriuretic peptide gene promoter activity, a marker of hypertrophy, in cultured neonatal rat ventricular myocytes. We have used a combination of transient transfection analysis and reverse transcriptase-polymerase chain reaction to examine the role of locally produced factors in contributing to this activation. Conditioned media from strained, but not static, cultures led to a dose-dependent increase in human brain natriuretic peptide gene promoter activity. This increase was completely blocked by losartan or BQ-123, implying a role for angiotensin and endothelin as autocrine/paracrine mediators of the response to strain. Inclusion of the same antagonists in the cultures themselves led to only partial inhibition (∼60%), whereas inclusion of exogenous endothelin or angiotensin II resulted in amplification of the strain response. Angiotensin II and endothelin appear to be arrayed in series in the regulatory circuitry; the angiotensin response was blocked by BQ-123, whereas the endothelin response was unaffected by losartan. Mechanical strain was also shown to stimulate expression of the endogenous angiotensinogen, angiotensin-converting enzyme, and endothelin genes in this system. Collectively, these data indicate that locally generated angiotensin II and endothelin, acting in series, play an important autocrine/paracrine role in mediating strain-dependent activation of cardiac-specific gene expression. Application of mechanical strain, or passive stretch, to myocardial cells in culture results in a series of phenotypic changes that closely resemble those that occur with myocyte hypertrophyin vivo. This includes activation of the immediate early gene family (e.g. c-fos, c-jun, c-myc, and egr-1), increased expression of the fetal gene program (e.g. atrial natriuretic peptide, α skeletal actin, and β-myosin heavy chain), and increased protein synthesis (1Komuro I. Katoh Y. Kaida T. Shibazaki Y. Kurabayashi M. Hoh E. Takaku F. Yazaki Y. J. Biol. Chem. 1991; 266: 1265-1268Abstract Full Text PDF PubMed Google Scholar, 2Sadoshima J. Jahn L. Takahashi T. Kulik T.J. Izumo S. J. Biol. Chem. 1992; 267: 10551-10560Abstract Full Text PDF PubMed Google Scholar). A number of recent studies have suggested that activation of autocrine/paracrine regulatory mechanisms in the myocardium may play an important, if not dominant, role in determining the myocyte response to hemodynamic load. Specific local regulators invoked as participating in this process include angiotensin II (AII) 1The abbreviations used are: AII, angiotensin II; RT-PCR, reverse transcriptase-polymerase chain reaction; BNP, brain natriuretic peptide; hBNP, human BNP; ET, endothelin; TGF-β, transforming growth factor β; AT1, angiotensin type 1; MAP, mitogen-activated protein; ACE, angiotensin-converting enzyme; bp, base pair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RAS, renin-angiotensin system; AOG, angiotensinogen. 1The abbreviations used are: AII, angiotensin II; RT-PCR, reverse transcriptase-polymerase chain reaction; BNP, brain natriuretic peptide; hBNP, human BNP; ET, endothelin; TGF-β, transforming growth factor β; AT1, angiotensin type 1; MAP, mitogen-activated protein; ACE, angiotensin-converting enzyme; bp, base pair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RAS, renin-angiotensin system; AOG, angiotensinogen. (3Sadoshima J. Xu Y. Slayter H.S. Izumo S. 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Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked strain-dependent increases in c-fos, α skeletal actin and atrial natriuretic factor gene expression in their system. Yamazaki et al. (4Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Mizuno T. Takano H. Hiroi Y. Ueki K. Tobe K. Kadowaki T. Nagai R. Yazaki Y. Circ. Res. 1995; 77: 258-265Crossref PubMed Scopus (264) Google Scholar, 5Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Mizuno T. Takano H. Hiroi Y. Ueki K. Tobe K. Kadowaki T. Nagai R. Yazaki Y. J. Clin. Invest. 1995; 96: 438-446Crossref PubMed Scopus (189) Google Scholar) confirmed that AII plays a significant role in determining the response to strain. In their hands, the induction of MAP kinase, MAP kinase kinase, Raf-1 kinase, or protein synthesis by strain was suppressed, although only by 50–70%, following blockade of the AT1 receptor (4Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Mizuno T. Takano H. Hiroi Y. Ueki K. Tobe K. Kadowaki T. Nagai R. Yazaki Y. Circ. Res. 1995; 77: 258-265Crossref PubMed Scopus (264) Google Scholar, 5Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Mizuno T. Takano H. Hiroi Y. Ueki K. Tobe K. Kadowaki T. Nagai R. Yazaki Y. J. Clin. Invest. 1995; 96: 438-446Crossref PubMed Scopus (189) Google Scholar). This subtotal inhibition implies variable dependence of different responses on locally generated AII. In an independent study, the same group found that the potent vasoconstrictor endothelin (ET) also participates as an intermediate in the MAP kinase response to strain (7Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). This effect was found to be additive with that produced by AII suggesting that the two operate in parallel as autocrine/paracrine effectors of hypertrophy. The cell culture studies have found corroboration in several whole animal models. Angiotensinogen, ACE, and renin gene expression have been identified in the heart (16Raman V.K. Lee Y.A. Lindpaintner K. Am. J. Cardiol. 1995; 76: 18D-23DAbstract Full Text PDF PubMed Scopus (53) Google Scholar, 17Baker K.M. Booz G.W. Dostal D.E. Annu. Rev. Physiol. 1992; 54: 227-241Crossref PubMed Scopus (494) Google Scholar) and shown, in selected models, to increase with application of mechanical strain in vitro (3Sadoshima J. Xu Y. Slayter H.S. Izumo S. Cell. 1993; 75: 977-984Abstract Full Text PDF PubMed Scopus (1162) Google Scholar,18Shyu K.G. Chen J.J. Shih N.L. Chang H. Wang D.L. Lien W.P. Liew C.C. Biochem. Biophy. Res. Commun. 1995; 211: 241-248Crossref PubMed Scopus (48) Google Scholar) or hemodynamic overload in vivo (19Baker K.M. Chernin M.I. Wixson S.K. Aceto J.F. Am. J. Physiol. 1990; 259: H324-H332PubMed Google Scholar, 20Schunkert H. Dzau V.J. Tang S.S. Hirsch A.T. Apstein C.S. 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Losartan, as completely blocked induction by AII. also blocked the AII blocked the losartan was in this This that AII and operate in appears that locally produced AII increases in leads to activation of the promoter and increased gene expression. AII has also been shown to increase of in cardiac J. Cell. Cardiol. 1995; Full Text PDF PubMed Scopus Google Scholar, A. A. Hypertension. 1994; PubMed Google Scholar), and to be a potent of those associated with hypertrophy (10Takahashi N. Calderone A. Izzo Jr., N.J. Maki T.M. Marsh J.D. Colucci W.S. J. Clin. Invest. 1994; 94: 1470-1476Crossref PubMed Scopus (177) Google Scholar, 11Parker T.G. Packer S.E. Schneider M.D. J. Clin. Invest. 1990; 85: 507-514Crossref PubMed Scopus (309) Google Scholar). locally generated an of autocrine/paracrine shown in of resulted in a in the of the gene promoter in the and the of the strain effect was The of the effect was confirmed by the of to reverse the that important for of promoter but not to of the response to mechanical strain. We mechanical strain to increased expression of the of or endothelin in this as a to activation of promoter activity, and if the of induction the model We used to a of angiotensinogen, ACE, and gene expression as a of following application of the strain shown in strain led to a in gene expression which was of culture in the The effect and by renin gene expression not be in these cultures that identified the renin gene in of rat not of ACE a and induction that with angiotensinogen. The effect was and of strain. induction of gene expression was following of strain, This effect of strain. The induction of gene expression in from activation of promoter activity. A of rat to a M. M. W. M. Hypertension. 1995; PubMed Google Scholar) was cultured that to mechanical strain. shown in strain a increase in activity. The strain-dependent induction was completely by losartan or the that AII from in the autocrine/paracrine In with this AII the promoter in these also a effect the of of promoter in this The an important role for AII and as autocrine/paracrine in the gene response to mechanical strain. of the to the strain response be to the of AII or in the culture this only of the implying the of an to gene promoter activation independent of autocrine/paracrine activity. for this from the that the strain to an independent effect in the of of AII and a number of the of an renin-angiotensin (16Raman V.K. Lee Y.A. Lindpaintner K. Am. J. Cardiol. 1995; 76: 18D-23DAbstract Full Text PDF PubMed Scopus (53) Google Scholar, 17Baker K.M. Booz G.W. Dostal D.E. Annu. Rev. Physiol. 1992; 54: 227-241Crossref PubMed Scopus (494) Google Scholar) in the and ACE found to be in these myocyte expression of was increased in the of and for following application of mechanical strain. and the response to strain, in in an This with the AII studies to a role for the cardiac in the autocrine/paracrine amplification of the strain response. We for expression of endogenous rat renin in these of the renin in from rat This in to of renin expression in the heart N. Shimoike H. Kinoshita M. Circulation. 1995; 92: 2690-2696Crossref PubMed Scopus (103) Google Scholar, 22Lee Y. Liang C. Lee M. Lindpaintner K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11035-11040Crossref PubMed Scopus (62) Google Scholar, 23Boer P.H. Ruzicka M. Lear W. Harmsen E. Rosenthal J. Leenen F.H.H. Am. J. Physiol. 1994; 267: H1630-H1636PubMed Google Scholar), and may in the of the for A of renin gene expression not the of the in the response to strain. from renin in vivo N. Circulation. 1994; PubMed Scopus Google Scholar) in the of vitro model used from in the used in the myocytes. of be produced in the or The studies a role for the AT1 and in the responses to AII and ET, and a role for and The that the of media completely by AT1 ACE or blockade implies that AII and operate a in the strain response. data this and indicate that the two are arrayed in series with AII to in leads to increased gene This with the by Yamazaki et al. (7Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) found that and in MAP kinase additive in their myocyte The not but that different regulatory may be in expression of of the the the by Yamazaki et al. (7Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. 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(7Yamazaki T. Komuro I. Kudoh S. Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), on the a of and gene expression in myocyte cultures to mechanical strain. In studies gene expression was by mechanical strain. The induction was of strain. this with the dependence AII in that and ACE gene expression increased a This dependence was by the that the strain-dependent induction of the gene promoter was completely with losartan or a was not with the gene implying that strain-dependent activation of the promoter the autocrine/paracrine of AII the culture studies not a role for mediators to in the AII in been shown to stimulate of J. Cell. Cardiol. 1995; Full Text PDF PubMed Scopus Google Scholar, A. A. Hypertension. 1994; PubMed Google Scholar), a in the myocardium (11Parker T.G. Packer S.E. Schneider M.D. J. Clin. 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Y. 1996; Scholar) We are to for with of the cells and to M. and C. F. for